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Aqueous extract of Thai medical Herbs (Phytoplex) InhibitsCell Proliferation and Induces Apoptosis in human cervical
cancer cell line (HeLa cells)
Somjit Chaiwattanarungruengpaisan1 Warunya Chakritbudsabong2,3
Nattapat Rutjanavate2 Rassameepen Phonarknguen1 Ganokon Urkasemsin2
Sasitorn Rungarunlert2*
1The Monitoring and Surveillance Center for Zoonotic Diseases in Wildlife and Exotic Animals (MoZWE), Faculty of Veterinary Science,
Mahidol University, Nakhon Pathom, Thailand2Department of Preclinic and Applied Animal Science, Faculty of Veterinary Science, Mahidol University, Nakhon Pathom, Thailand
3Department of Clinical Science and Public Health, Faculty of Veterinary Science, Mahidol University, Nakhon Pathom, Thailand
*Corresponding author, E-mail address: [email protected]
Abstract
Cervical cancer remains a leading cause of cancer mortality in females. Chemotherapy is important as it
was a part of the main treatment for this type of cancer. However, multidrug resistance and serious side effects
have been major problems in cervical cancer chemotherapy. Therefore, the search for new anticancer drugs
from the native medicinal herbs, is very attractive. The combination extract of eight Thai medicinal herb recipes,
namely Phytoplex, is a commercial product of The Government Pharmaceutical Organization, has anti-cancer
effect on hepatocellular carcinoma cells (HepG2) in vitro. However, the anticancer effects and molecular
mechanisms of Phytoplex on cervical cancer have not yet been studied. The aim of this study was to evaluate
the inhibitory effect of Phytoplex on human cervical adenocarcinoma cells (HeLa cells) in vitro. HeLa cells were
treated with low concentrations of Phytoplex (50, 100, 500, 1,000 μg/ml) and high concentrations of Phytoplex
(2,000 and 5,000 μg/ml) compared with a positive control (0.1% mitomycin C) and a negative control (0 μg/ml of
Phytoplex) for 24, 48 and 72 h. Then, cell viability was evaluated using an MTT assay. The activities of caspase-3
(apoptosis marker) and Ki-67 (proliferation marker)†were investigated using an immunofluorescence assay. At
24 h, lower concentrations of Phytoplex promoted cell viability, while higher concentrations inhibited cell viability
(P < 0.05). In addition, the inhibitory effect of Phytoplex continuously increased from 24 to 72 h of incubation
peroid at high concentrations of Phytoplex. Moreover, Phytoplex inhibited HeLa cell proliferation with an IC50
value of 1,972.43, 1,230.10 and 1,317.67 μg/ml at 24, 48 and 72 h, respectively. The activity of caspase-3 of
HeLa cells treated with high concentration of Phytoplex seemed to be higher than of negative control (0 μg/ml).
Activity of Ki-67 was higher in lower concentrations of Phytoplex, than that in higher concentrations of
Phytoplex. Therefore, HeLa cell growth inhibition was dose-and time-dependent. The study suggested that high
concentrations (2,000 and 5,000 μg/ml) of Phytoplex exerted inhibitory effect in HeLa cell growth by inducing
apoptosis via activation of caspase-3.
Keywords: cervical cancer, Phytoplex, HeLa cells, herbs, viability
Journal of Applied Animal Science 2018; 11(2): 31-44.
Research Articles
32 Journal of Applied Animal Science Vol.11 No.2 May-August 2018
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«—¥Õ—μ√“√Õ¥™’«‘μ¢Õ߇´≈≈å‚¥¬«‘∏’ MTT μ√«®°“√· ¥ßÕÕ°¢Õß caspase-3 (∫àß∫Õ°°“√쓬¢Õ߇´≈≈å) ·≈– Ki-67 (∫àß∫Õ°
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Journal of Applied Animal Science 2018; 11(2): 31-44.
Journal of Applied Animal Science Vol.11 No.2 May-August 2018 33
Introduction
In human, cervical cancer, a Papillomavirus
infection-related cancer, was the fourth most frequent
cancer globally found among women during 2012, with
age-standardized incidence rate (ASRs) of 14 per 100,000
(an estimated 528,000 new cases diagnosed annually).
During 2012, the worldwide number of deaths from
cervical cancer was 265,672 heads, accounting for 7.5%
of all cancer in women (Serrano et al., 2018). Even
though the incidence of cervical cancer has decreased in
developed countries, this cancer is a serious public health
as the second leading cause of female cancer in Thailand,
with ASRs of 17.8 per 100,000 (about 8,184 new cases
diagnosed annually) (Bruni et al., 2017). Chemotherapy
is considered the main treatment of cervical cancer.
However, it contributes to several serious side effects,
including alopecia, nausea and vomiting, peripheral
neuropathy and fatigue (Sun et al., 2014). Furthermore,
numerous patients with recurrent cervical cancer do not
show any response to the chemotherapy because
of multidrug resistance (Zhu et al., 2016). Hence, the
discovery and development of new chemotherapeutic
drugs is urgently necessitated.
Medicinal herb is a medical system utilizing
plants, their extracts, or their recipes to promote health
status as well as to prevent and cure diseases for thousand
years. In addition, synergistic effects of medicinal herb
recipes has been reported (Che et al. 2013). In Thailand,
traditional medicinal herb recipes have been incrementally
used as alternative treatments for anticancer drugs due to
low cost and low risk of side effects (Poonthananiwatkul
et al., 2015). Besides, thai medicinal herb recipes do not
only possess anticancer activities in several human cancer
cell lines, but they also improve survival rate and quality
of life of cancer patients when they are used for adjuvant
therapy with traditional chemotherapeutic drugs (Thisoda
et al., 2013).
Phytoplex, one of the most outstanding Thai
medicinal herb recipes, is a commercial product launched
by the Government Pharmaceutical Organization (GPO)
of Thailand, which has been ordered for curing patients
with cancer by Dr. Sommai Thongprasert (traditional Thai
medicine practitioner) (Akaraserenont et al., 1999).
Phytoplex is a combination of eight plants: Acanthus
ebracteatus (Ngueak-Pla-Moo, Acanthaceae), Ammannia
baccifera (Ma-Fai-Duen-Haa, Lythraceae), Canna indica
(Put-Ta-Rak-Sa, Cannaceae), Clinacanthus nutans
(Pha-Ya-Yor, Acanthaceae), Mallotus philippensis
(Tang-tuay, Euphorbiaceae), Polygala chinensis (Peek-Kai-
Dam, Polygalaceae), Premna herbacea (Khao-Yen-Tai,
Lamiaceae), and Smilax corbularia (Khao-Yen-Nuea,
Smilacaceae). The previous study reported that the use of
anticancer properties of a single herbal medicine could
induced apoptosis in cancer cells by active ingredient in
each plant of Phytoplex including β-sitosterol in A.
ebracteatus (Awad et al., 2003), triterpenes and coumarins
in A. baccifera (Loganayaki et al., 2012), stigmasterol and
6 beta-hydroxystigmasta-4, 22-diene-3-one in C. indica
(Darsini et al., 2015), stigmasterol in C. nutans (Ng et al.,
2017), rottlerin in M. philippensis (Gangwar et al., 2014),
terpenoid in P. chinensis and P. herbacea (Yang and Dou,
2010), and quercetin in S. corbularia (Granado-Serrano
et al., 2006). However, there was no report of anticancer
properties of combine use of these plants. Moreover,
34 Journal of Applied Animal Science Vol.11 No.2 May-August 2018
earlier studies have indicated that Phytoplex inhibited
vascular endothelial cell proliferation in human umbilical
vein endothelial cells (HUVECs) (Akaraserenont et al.,
1999) and supressed the growth of hepatocellular
carcinoma cells (HepG2) transplanted in mice due to
tumor anti-angiogenesis effect (Duansak et al., 2007).
Besides, the chronic toxicity of Phytoplex demonstrated
that the oral administration with Phytoplex of 0.24, 1.2,
3.6 g/kg/day for six months did not affect both health sta-
tus and body weight in Wistar rats (Chivapat et al., 2010).
However, the anticancer effects and actual molecular
mechanisms of Phytoplex on cervical cancer have not yet
been studied. Accordingly, the aim of this study was to
evaluate the effect of Phytoplex on growth inhibition of
human cervical adenocarcinoma cells (HeLa cells) in vitro,
which could be developed to the adjuvant anticancer
therapeutic agent of cervical cancer in human in the
future.
Materials and methods
Chemicals, reagents and culture media
All chemicals and reagents were purchased from
Sigma-Aldrich Co. (Saint-Louis, MO, USA) and cell
culture products were purchased from GIBCO (Carlsbad,
CA, USA).
Cell culture
HeLa cells (ATCC® CCL-2) were kindly provided
by the Monitoring and Surveillance Center for Zoonotic
Diseases in Wildlife and Exotic Animals (MoZWE),
passaged every 3-4 days with trypsin/EDTA and cultured
in a fibroblast medium (FM) containing Dulbecco's
modified Eagle medium (DMEM), 10% fetal bovine
serum (FBS), 1% glutamax-L and 1% penicillin-
streptomycin. Cells were incubated in a humidified 37oC,
5% CO2 incubator.
Herbal extract and treatments
Phytoplex (GPO, Bangkok, Thailand), an aqueous
crude extract of eight plants packed in a capsule, was
extracted with phosphate buffered saline (PBS) at 100oC
for 2 h. Stock solution of Phytoplex was 10 mg/ml and
stored at 4oC, meanwhile further dilutions were prepared
in FM. Hela cells were treated with low concentrations
of Phytoplex (50, 100, 500, 1,000 μg/ml) and high
concentrations of Phytoplex (2,000 and 5,000 μg/ml). The
cells treated with 0.1% mitomycin C (5 μg/ml) were used
as positive control, and untreated cells (0 μg/ml of
Phytoplex) were considered negative control.
Morphological observation
HeLa cells were seeded into 6-well plates with 2 x
105 cells/well. After 6 h of incubation, the medium was
changed with different concentrations of Phytoplex.
After 24, 48 and 72 h of incubation, cell morphology was
examined under an inverted microscope. Images were
captured by an Axiovert 40 CFL microscope connected
to AxioCam 105 color camera (Carl Zeiss, Germany).
Cell viability assay
MTT assay was applied to measure cytotoxic
effect of Phytoplex on HeLa cells, as already described
by Berenyi et al. (2013). Briefly, cells were applied into a
96-well plate with a density of 5 x 103 cells/well. After 6 h
Journal of Applied Animal Science Vol.11 No.2 May-August 2018 35
of adherence, the medium was changed with various
concentrations of Phytoplex. After 24, 48 and 72 h, 20 μl
of 3-(4, 5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium
bromide (MTT) solution at 5 mg/ml in PBS was added to
each well and incubated for 3 h. Then, the media were
replaced with 100 μl of Dimethyl sulfoxide (DMSO) per
well to dissolve the formation of formazan crystals by the
mitochondrial dehydrogenase activity in the living cells.
Each drug concentration was assayed in 8 wells and
repeated at least three times. The absorbance values were
measured by an ELX808 automatic microplate reader
(BioTek Inc., Winooski, VT, USA) at the wavelength of
450 nm. The Hela cell viability was calculated as a
percentage from the equation shown below. The IC50
value (drug concentration inhibiting cell proliferation by
50%) was determined from linear regression analysis.
The percentage of HeLa cell viability was
calculated using the formula as followed:
Viability (%) = (Mean OD of test sample-Mean OD of the blank)
(Mean OD of the control-Mean OD of the blank)
Detection of apoptosis and proliferation by
immunofluorescence staining
HeLa cells were applied into a 24-well plate at a
density of 2 x 104 cells/well. After 24, 48 and 72 h of
treatment, they were fixed in 4% paraformaldehyde for 15
min, permeabilized with 0.25% Triton-X100 for 10 min
and blocked with 1% bovine serum albumin in PBS.
The fixed cells were stained with caspase-3 antibody
(sc-7148, diluted 1:200; Santa Cruz Biotechnology, USA)
to investigate the apoptotic activity, and stained with
Ki-67 antibody (sc-15402, diluted 1:200; Santa Cruz
Biotechnology, USA) to evaluate the cell proliferation at
room temperature for 1 h. Then, the samples were labeled
with AlexaFluor® 594 goat anti-rabbit IgG (A11037,
diluted 1:2000; Invitrogen Life Technologies, USA) at
room temperature for 1 h.
The stained cells were mounted onto slide with 4',
6-diamidino-2-phenylindole (DAPI) staining (Vectashield®
H-1200, Vector Laboratories, UK) and assessed under a
fluorescence microscope. Images were captured using an
Axioskop 40 microscope connected to AxioCam MRc
camera (Carl Zeiss, Germany).
Statistical analysis
Each experiment was repeated three times. Mean
data values were presented with their deviation (mean ±
SD). Statistical analyses were performed according to
the SPSS statistics version 18.0 (SPSS, 2010). Analysis
of variance (ANOVA) was performed by Dunnett's T3
test. A value of P < 0.05 was considered statistically
significant.
Results
Cell morphology
Under the inverted phase contrast microscope, no
typical change in morphology was observed from cells
treated with Phytoplex at low concentrations (50, 100, 500
and 1,000 μg/ml) compared with untreated cells at 24, 48
and 72 h (data not shown). The cells were well spread and
appeared in regular polygonal shape with a few round
cells. In Figure 1, HeLa cells treated with Phytoplex at
36 Journal of Applied Animal Science Vol.11 No.2 May-August 2018
high concentrations (2,000 and 5,000 μg/ml) showed the
decrease in cell volume and the obviously morphological
changes including cellular shrinkage, rounding and
vacuole formation. Moreover, cells detached from the
surface and cell debris were also detected.
Cell viability
According to the MTT test, Figure 2 demonstrates
that the inhibition of HeLa cell growth was drug
concentration- and time-dependent. The maximum growth
inhibitory effect of Phytoplex was observed at high
concentrations (2,000 and 5,000 μg/ml). The percentage
of viable cells treated with 2,000 μg/ml of Phytoplex,
compared to the negative control (0 μg/ml of Phytoplex),
significantly decreased from 48.3 ± 12.92% after 24 h
to 16.2 ± 3.46 % and 13.4 ± 4.49 % by 48 and 72 h,
respectively (P-value < 0.05). Furthermore, a strong
diminishment of viable cell number was appeared by 24 h
of Phytoplex treatment at 5,000 μg/ml. Moreover, the
complete death of viable cells was noticed by 72 h.
According to Phytoplex treatment at high concentrations
(2,000 and 5,000 μg/ml) significant reduction in cell
viability was shown compared to positive control (0.1%
mitomycin C) at different points of treatment (24, 48 and
72 h). Furthermore, Phytoplex inhibited the proliferation
of HeLa cells with an IC50
value of 1,972.43, 1,230.10 and
1,317.67 μg/ml at 24, 48 and 72 h, respectively. However,
the low concentrations of Phytoplex (50, 100, 500, 1,000
μg/ml) promoted cell growth compared with untreated
cells at 24 h.
Cell apoptosis
Caspase-3 in HeLa cells were stained in red with
caspase-3 antibody, while the nuclei were counterstained
in blue with DAPI (Figure 3). The activity of caspase-3
seemed to be higher in HeLa cells treated with high
concentrations of Phytoplex (2,000 and 5,000 μg/ml)
and positive controls (0.1% mitomycin C) than in
negative controls (0 μg/ml of Phytoplex). The higher the
concentration of Phytoplex, the more the decrease in
cell number was found. Moreover, under continuous
exposure to Phytoplex, an increased numbers of cells
positive to caspase-3 staining was observed, indicating
the progression of apoptosis across the cell population.
Cell proliferation
Ki-67, a nuclear protein, was stained in red and the
nucleus was counterstained in blue with DAPI (Figure 4).
Activity of Ki-67 was high in HeLa cells treated with low
concentrations (50, 100, 500 and 1,000 μg/ml) of Phytoplex
and negative control, whereas low activity was found in
those treated with high concentrations (2,000 and 5,000
μg/ml) of Phytoplex.
Discussion
Cervical cancer has been one of the most frequent
cancers causing mortality among females in Thailand and
USA (National cancer institute, 2012). As cancer, an
induction of cell death using cytotoxic agents such as
chemotherapy was important strategies of treatment.
Recently, the resistance of anticancer synthetic drug was
reported worldwide (Mohammad et al., 2015). Therefore,
the discoveries of new and safe drugs from native
Journal of Applied Animal Science Vol.11 No.2 May-August 2018 37
medicinal herbs for cervical cancer treatment have become
necessary. In Thailand, Phytoplex was produced as a
commercial drug containing the extracts from eight Thai
medicinal herbs. Currently, anti-angiogenesis effect on
HUVECs and HepG2 cells was reported (Akaraserenont
et al., 1999; Duansak, 2007). Thus, it is important to
investigate the more benefits of this drug on other
cencer cells as well as its anticancer mechanism. In this
study, we demonstrated that aqueous extract of Phytoplex
exhibited a dose- and time-dependant activity, having
IC50
value of 1,972.43, 1,230.10 and 1,317.67 μg/ml at 24,
48 and 72 h, respectively.
Apoptosis (programmed cell death) has been one
of the reliable indicators for evaluating cytotoxic agents.
It could be characterized by cell rounding and shrinkage,
nuclear condensation and fragmentation, plasma membrane
blebbing and reduced cell volume (Safarzadeh et al., 2014).
Caspase-3, one of the key executioners of apoptosis, could
be used to determine mechanisms of apoptosis in cellular
assays (Elmore, 2007). In this study, we demonstrated that
HeLa cells treated with high concentrations of Phytoplex
(2,000 and 5,000 μg/ml) exhibited morphological
deformation, while the proliferation was inhibited due to
cell apoptosis. This was confirmed by the increase in
caspase-3 activity within 24, 48 and 72 h after Phytoplex
treatment via immunofluorescence assay. Therefore, we
revealed that the main molecular mechanisms of
Phytoplex in Hela cells were to inhibit cell proliferation
and induce apoptosis through caspase-3 activation. This
finding was supported by the previous study. It revealed
that β-sitosterol in A. ebracteatus induced cell apoptosis
in human breast cancer cell line (MDA-MB-231) via
caspase-3 activation (Awad et al., 2003). In addition, this
active ingredient possessed a number of anticancer
effects, such as increased caspase-3 activation, decreased
antiapoptotic Bcl-2 expression and increased proapoptotic
Bax expression in human colon cancer cells (HT116)
(Choi et al., 2003). Moreover, A. ebracteatus could inhibit
cervical cancer growth, vascular endothelial growth
factor expression and angiogenesis in a CaSki-cell
transplanted in mice (Mahasiripanth et al., 2012). Besides,
the triterpenes and coumarins in A. baccifera, that extracted
by using methanol, exerted cytotoxicity against HeLa
cells in vitro, but no cytotoxic effects were observed
against NIH/3T3, a normal cell line (Loganayaki et al.,
2012). The mechanism of triterpenes induced apoptosis
in cancer cells via the activation of caspase-8 and -9 (Uto
et al., 2013). Coumarin induced cell cycle arrest and
apoptosis in Hela cell by inducing internucleosomal
fragmentation of DNA and reducing mitochondrial
membrane potential. Also, coumarin increased the
expression of the Bax (pro-apoptosis protein), promoted
the release of cytochrome c and activated caspase 3
(Chuang et al. 2007). In addition, the former study
indicated that stigmasterol and 6 beta-hydroxystigmasta-
4, 22-diene-3-one in C. indica exhibited cytotoxicity
activity against P388 leukemia cells (ED50
= 55.50 and
37.50 μg/ml) (Darsini et al., 2015). Moreover,
stigmasterol in C. nutans had antiproliferative effects on
HeLa cells but significant cytotoxic activities were
observed against Vero, normal kidney cell line (Zakaria
et al., 2017). Stigmasterol induced intracellular reactive
oxygen species (ROS) generation and induced cell
apoptosis through intrinsic and extrinsic caspase pathways
38 Journal of Applied Animal Science Vol.11 No.2 May-August 2018
(Ng et al., 2017). Rottlerin in M. philippensis inhibited
histamine-induced H1-receptor gene in HeLa cells
(Gangwar et al., 2014). According to in vivo studies,
treatments of Daltonís ascites lymphoma-mice with single
herb, including P. chinensis and P. herbacea, contributed
to enhance average life span and reduced tumor xenograft
volume compared with the control group (Alagammal
et al., 2012). Terpenoid in P. chinensis and P. herbacea
drive up-regulation of Bax and down-regulation of Bcl-2
(anti-apoptotic protein), resulting in cytochrome c release,
caspase activation and apoptotic cell death (Yang and
Dou, 2010). Quercetin in S. corbularia induced apoptosis
in HepG2 by the activation of caspase-3 and -9, the higher
expression of pro-apoptotic Bcl-s family members (Bsl-
xs and Bax) and the lower level of anti-apoptotic (Bcl-
xL) (Granado-Serrano et al., 2006). Mitomycin C, an
anticancer drug used for blocking DNA and RNA
replications and stopping protein synthesis, led to an
inhibition of cell mitosis (Sartorelli et al., 1994). In this
study, mitomycin C was used as a positive control for
apoptosis examination because it induced apoptosis in
Hela cells and human breast cancer cell line (MCF-7)
via a caspase-3 (Pirnia et al., 2002; Cheng et al., 2009).
Ki-67, a marker of cancer proliferation, was
associated with aggressive cancer. We demonstrated that
HeLa cell proliferation was inhibited when being treated
with high concentrations of Phytoplex (2,000 and 5,000
μg/ml) , as confirmed by immunofluorescence assay.
The use of multiple herb extracts against cancer in
complex recipes provided better efficicay than single
active ingredient or herb at the equivalent dose (or
concentration). This combination resulted in chemical
combination effects. The main mechanisms are
hypothesized to be either potentiated or prolonged, and/
or its adverse effects reduced, because of synergistic or
antagonistic effects, by adding of other herbs (Kiyohara
et al., 2004; Zhou et al., 2016). The former study
demonstrated that the aqueous extract of A. ebracteatus
alone had weak antiproliferative effect on HeLa cells as
seen from IC50 value of 6,072.50 μg/ml (Mahasiripanth
et al., 2012), which was lower than that from Phytoplex
in the current study (1,230.10 μg/ml at 48 h incubation
period). Moreover, the aqueous extract of M. philippensis
at 100 μg/ml alone had no antiproliferative effect to
Hela (Sharma and Varma, 2011). On the other hand, the
use of single herb either the aqueous extract of C. nutans
or the methanol extract of A. baccifera had antiproliferative
effect against HeLa cells (IC50=13 and 130 μg/ml,
respectively), which was lower than that of Phytoplex
(Yusmazura et al., 2017). Aqueous extract of S. corbalaria
alone possessed low cytotoxic activity against human
colon adenocarcinoma (LS-174T) and human large cell
lung carcinoma cell lines (COR-L23), their percentages
of cell viability for 50 μg/ml concentration after 72 h
incubation were 60.1 and 98.9, respectively (Itharat et al.,
2004). This difference in IC50
could be explained that the
combined use contributed to synergistic or antagonistic
effects of anticancer (Pal et al., 2003), the type of solvents
for herbal extraction and the sensitivity of various kinds
of cancer cell lines to anticancer herbs (Sharma and
Varma, 2011). Here, we demonstrated that Phytoplex has
chemical combination effects, synergistic or antogonistic
effects, against Hela cells.
Further investigations for the mechanisms of Phytoplex
Journal of Applied Animal Science Vol.11 No.2 May-August 2018 39
on HeLa cells are needed including an alteration in cell
cycle, an immunomodulatory effect and an activation of
autophagic activity. Moreover, studies in animal model
should be conducted to assure the efficacy and safety so
that Phytoplex would be the adjuvant anticancer therapeu-
tic agent of cervical cancer in human in the future.
Figure 1. The morphology of HeLa cells treated with different concentrations of Phytoplex (1,000, 2,000 and 5,000 μg/
ml) after 24, 48 and 72 h, compared to the negative control (0 μg/ml of Phytoplex) and the positive control (0.1%
Mitomycin C). Magnification: 10x.
40 Journal of Applied Animal Science Vol.11 No.2 May-August 2018
Figure 2. The percentage of cell viability after being treated with different concentrations of Phytoplex (1,000, 2,000 and
5,000 μg/ml) after 24, 48 and 72 h, compared to the negative control (0 μg/ml of Phytoplex) and the positive control (0.1%
Mitomycin C). Different letters demonstrates statistical significance (P-value < 0.05, Dunnett's T3 test).
Figure 4. HeLa cells processed for immunofluorescence assay of Ki-67 antibody (red) and counterstained with DAPI
(blue). Magnification: 40x.
Figure 3. HeLa cells processed for immunofluorescence assay of caspase-3 antibody (red) and counterstained with DAPI
(blue). Magnification: 40x.
Journal of Applied Animal Science Vol.11 No.2 May-August 2018 41
Conclusion
Our study revealed anticancer properties of
Phytoplex against HeLa cells. Phytoplex exhibits
numerous anti-cancer effects including deformation of
morphology, inhibition of cell growth, suppression of
Ki-67 and induction of apoptosis via activation of caspase-
3. Moreover, only high concentrations (2,000 and 5,000
μg/ml) of Phytoplex exerted growth inhibitory effect in
HeLa cells. Hence, HeLa cell growth inhibition was a
dose- and time-dependent manner.
Acknowledgments
This study was financially supported by grants from
Mahidol University and Faculty of Veterinary Science,
Mahidol University, Thailand. We would like to thank the
following researchers for their contributions: Sookruetai
Boonmasawai for the gift of MTT, Dr. Panpanga Sangsuriya
for kindhearted statistical analysis, and Assistant
Professor Atthaporn Roongsitthichai for language
scrutinization.
References
Akaraserenont P, Wongkajornsilp A, Chotewuttakorn S,
Thaworn A, Haubpresert S, Kraisintu K. The effect
of herbal extracts (GPO 1986) on proliferation of
vascular endothelium. Thai J Pharmacol. 1999; 21:
205-212.
Alagammal M, Nishanthini A, Mohan VR. Anticancer
activity of the whole plant of ethanolextract of
Polygala chinensis l. (Polygalaceae). J Curr Chem
Pharm Sc. 2012; 2(4): 299-305.
Awad AB, Roy R, Fink CS. β-sitosterol, a plant sterol,
induces apoptosis and activates key caspases in
MDA-MB-231 human breast cancer cells. Oncol
Rep. 2003; 10(2): 497-500.
Berényi A, Minorics R, Iv nyi Z, Ocsovszki I, Ducza E,
Thole H, et al. Synthesis and investigation of the
anticancer effects of estrone-16-oxime ethers in vitro.
Steroids. 2013; 78(1): 69-78.
Bruni L, Barrionuevo-Rosas L, Albero G, Serrano B, Mena
M, GÓmez D, et al. Human papillomavirus and
related diseases report [Internet]. ICO Information
Centre on HPV and Cancer (HPV Information
Centre). 2017 [cited 2017 Aug 1]. Available from:
http://www.hpvcentre.net/statistics/reports/THA.
pdf.
Che CT, Wang ZJ Chow MSS, Lam CWK. Herb-herb
combination for therapeutic enhancement and
advancement: theory, practice and future perspectives.
Molecules. 2013; 18: 5125-5141.
Cheng MH, Cheng HT, Lin SS, Young SC, Pai CJ, Liao
PH, et al. Apoptotic death mode of mitomycin
C-treated HeLa cells and cellular localization of
mitomycin C-induced P-glycoprotein. Drug Chem
Toxicol. 2009; 32(2): 158-168.
Chivapat S, Attawish A, Chavalittumrong P,
Chantarateptawan V. Chronic toxicity of GPO 1986
herbal extract. J Thai Trad Alter Med. 2010; 8(1):
15-28.
42 Journal of Applied Animal Science Vol.11 No.2 May-August 2018
Choi YH, Kong KR, Kim Y, Jung K, Ki J, Rhee S, et al.
Induction of Bax and activation of caspases during
β-sitosterol-mediated apoptosis in human colon
cancer cells. Int J Oncol. 2003; 23(6): 1657-1662.
Chuang JY, Huang YF, Lu HF, Ho HC, Yang JS, Li TM.
Coumarin induces cell cycle arrest and apoptosis
in human cervical cancer Hela cells through a
amitochondria- and caspase-3 dependent mechanism
and NF-κ down-regulation. In Vivo. 2007; 21:
1003-1010.
Darsini AIP, Shamshad S, Paul MJ. Canna indica (L.): A
plant with potential healing power: a review. Int J
Pharm Bio Sci. 2015; 6(2): 1-8.
Duansak S. Effects of herbal extract (GPO 1986) on tumor
angiogenesis of hepatocellular carcinoma cells
(HepG2) implanted in nude mice. PhD Thesis.
Graduate School, Chulalongkorn University. 2007.
Elmore S. Apoptosis: A review of programmed cell death.
Toxicol Pathol. 2007; 35(4): 495-516.
Gangwar M, Goel RK, Nath G. Mallotus philippinensis
Muell. Arg (Euphorbiaceae): ethnopharmacology
and phytochemistry Review. Biomed Res Int.
2014; 1-13.
Granado-Serrano AB, Martin MA, Bravo L, Goya L,
Ramos S. Quercetin induces apoptosis via caspase
activation, regulation of Bcl-2, and inhibition of PI-
3-kinase/Akt and ERK pathway in human hepatoma
cell line (HepG2). J Nutr. 2006; 136(11): 2715-2721.
Itharat A, Houghton PJ, Eno-Amooquaye E, Burke PJ,
Sampson JH, Raman A. In vitro cytotoxic activity of
Thai medicinal plants used traditionally to treat
cancer. J Ethnopharmacol. 2004; 90(1): 33-38.
Kiyohara H, Matsumoto T, Yamada H. Combination
effects of herbs in a multi-herbal formula: expression
of Juzen-taiho-to's immuno-modulatory activity
on the intestinal immune system. Evid Based
Complement Alternat Med. 2004; 1(1): 83-91.
Loganayaki N, Manian S. Antitumor activity of the
methanolic extract of Ammannia baccifera L. against
Dalton's ascites lymphoma induced ascitic and
solid tumors in mice. J Ethnopharmacol. 2012; 142(1):
305-309.
Mahasiripanth T, Hokputsa S, Niruthisard S, Bhattarakosol
P, Patumraj S. Effects of Acanthus ebracteatus Vahl
on tumor angiogenesis and on tumor growth in nude
mice implanted with cervical cancer. Cancer Manag
Res. 2012; 4: 269-279.
Mohammad RM, Muqbil I, Lowe L, Yedjou C, Hsu HY,
Lin LZ, et al. Broad targeting of resistance to
apoptosis in cancer. Semin Cancer Biol. 2015;
35(0): 78-103.
Journal of Applied Animal Science Vol.11 No.2 May-August 2018 43
National cancer institute. Hospital-based cancer registry
annual report 2012. Information technology
division, National cancer institute, Department of
medical services, Ministry of public health, Thailand;
2012. p.1-59.
Ng PY, Chye SM, Ng CH, Koh RY, Tiong YL, Pui LP.
Clinacanthus nutans hexane extracts induce
apoptosis through a caspase-dependent pathway in
human cancer cell lines. Asian Pac J Cancer Prev.
2017; 18: 917.
Pal SK, Shukla Y. Herbal medicine: current status and
the future. Asian Pac J Cancer Prev. 2003; 4(4):
281-288.
Pirnia F, Schneider E, Betticher DC, Borner MM.
Mitomycin C induces apoptosis and caspase-8 and -9
processing through a caspase-3 and Fas-independent
pathway. Cell Death Differ. 2002; 9(9): 905-914.
Poonthananiwatkul B, Lim RH, Howard RL, Pibanpaknitee
P, Williamson EM. Traditional medicine use by
cancer patients in Thailand. J Ethnopharmacol. 2015;
168: 100-107.
Safarzadeh E, Sandoghchian SS, Baradaran B. Herbal
medicine as inducers of apoptosis in cancer
treatment. Adv Pharm Bull. 2014; 4: 421-427.
Sartorelli AC, Hodnick WF, Belcourt MF, Tomasz M,
Haffty B, Fischer JJ, et al. Mitomycin C: a prototype
bioreductive agent. Oncol Res. 1994; 6(10-11):
501-508.
Serrano B, Brotons M, Bosch FX, Bruni L.
Epidemiology and burden of HPV-related
disease. Best Pract Res Clin Obstet Gynaecol.
2018; 47: 14-26.
Sharma J, Varma R. A review on endangered plant of
Mallotus philippensis (Lam.) M.Arg. Pharmaco
onl. 2011; 3: 1256-1265.
Sun C, Brown AJ, Jhingran A, Frumovitz M, Ramondetta
L, Bodurka DC. Patient preferences for side effects
associated with cervical cancer treatment. Int
J Gynecol Cancer. 2014; 24(6): 1077-1084.
Thisoda P, Ketsa-Ard K, Thongprasert S, Vongsakul M,
Picha P, Karbwang J, et al. Immunostimulating
effect of a well-known Thai folkloric remedy in
breast cancer patients. Asian Pac J Cancer Prev.
2013; 14(4): 2599-2605.
Uto T, Sakamoto A, Tung AH, Fujiki T, Kishihara K, Oiso
S, et al. Anti-proliferative activities and apoptosis
induction by triterpenes derived from Eriobotrya
japonica in human leukemia cell lines. Int J Mol
Sci. 2013; 14(2): 4106-4120.
44 Journal of Applied Animal Science Vol.11 No.2 May-August 2018
Yang H, Dou GP. Targeting apoptosis pathway with
natural terpenoids: implications for treatment of
breast and prostate cancer. Curr Drug Targets. 2010;
11(6): 433-744.
Yumazura Z, Lim WY, Fakhuruddin N. Anti-cancer effects
of Clinacanthus nutans extract towards human
cervical cancer cell line, Hela. J of Biomed & Clin
Sci. 2017; 2(1): 11-19.
Zakaria Y, Yee LW, Hassan NF. Anti-cancer effects of
Clinacanthus nutans extract towards human
cervical cancer cell line, Hela. J Biomed Clin Sci.
2017; 2(1): 11-19.
Zhou X, Seto SW, Chang D, Kiat H, Razmovski-
Naumovski V, Chan K, et al. Synergistic effects of
Chinese herbal medicine: a comprehensive review
of methodology and current research. Front
Pharmacol. 2016; 7: 201.
Zhu H, Luo H, Zhang W, Shen Z, Hu X, Zhu X. Molecular
mechanisms of cisplatin resistance in cervical
cancer. Drug Des Devel Ther. 2016; 10: 1885-1895.
